The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication, and to use this knowledge to design broad-spectrum quorum sensing antagonists with potential therapeutic uses. Here we propose a cross-disciplinary investigation of LuxR-type quorum-sensing receptors from two human pathogens, Chromobacterium violaceum and Pseudomonas aeruginosa. We propose to combine synthetic organic chemistry, bacterial genetics, biochemistry and x-ray crystallography to identify and characterize signaling antagonists; these antagonists will serve as lead compounds for the development of antibacterial drugs designed to modulate quorum sensing. In our first aim, we will use high-throughput screening and in vivo assays to identify novel quorum-sensing antagonists active in C. violaceum. In our second aim, we will investigate the mechanisms by which these antagonists function using biochemical assays and x-ray crystallography. This work draws upon extensive preliminary data and provides a foundation for efforts to optimize the antagonists discovered in the first aim. The third aim extends the scope of this work to the clinically important bacterium P. aeruginosa. We propose a similar array of approaches to identify and characterize antagonists of its LuxR-type quorum-sensing receptor. Potent antagonists will be evaluated in a mouse infection assay with the aim of moving forward lead molecules for development into novel anti-bacterial therapeutics.